Nucleotide sequences which hybridize specifically with bacterial strains of the mycobacterium avium-intracellulare complex

ABSTRACT

A nucleic acid sequence which specifically hybridizes with a genomic nucleic acid sequence of strains of the complex Mycobacterium avium-intracellulare selected from nucleotide sequence SEQ ID No 1, nucleotide sequence SEQ ID No 2, complementary sequences thereof, and sequences differing therefrom by the mutation, insertion, deletion or substitution of one or more bases, but does not hybridize with the nucleic acids of mycobacteria which do not belong to said complex. Fragments of the sequence may be used as primers for amplifying sequences which are specific for strains of the complex Mycobacterium avium-intracellulare, and as nucleic probes which are specific for nucleic sequences of strains of the complex Mycobacterium avium-intracellulare. A method for detecting strains of the complex Mycobacterium avium-intracellulare in a biological sample, and a kit for implementing the method, are also provided.

BACKGROUND OF THE INVENTION

(i) Field of the Invention

The present invention relates to a nucleic sequence specific for theMycobacterium avium-intracellulare complex, as well as particularfragments of this sequence, which are capable of acting as nucleicprobes for the detection of a nucleic acid specific for strainsbelonging to the Mycobacterium avium-intracellulare complex or asnucleic primers in the amplification of DNA or RNA obtained from strainsof the Mycobacterium avium-intracellulare complex in a biologicalsample.

The invention also relates to a method of detection of strains belongingto the Mycobacterium avium-intracellulare complex in a biological sampleusing nucleic acid fragments, as well as a kit for implementing thismethod.

(ii) Description of Related Art

The number of infections caused by opportunistic mycobacteria isincreasing, partly because they are frequent in AIDS patients. Accordingto bacteriological studies carried out over the past few years, the mostimportant opportunistic bacteria are: M. kansasii, M. xenopi, M. simiaeand especially mycobacteria belonging to the complex: M.avium-intracellulare (MAI complex). However, geographical variationsexist in the incidence of the infection by these various opportunisticmycobacteria.

In a recent study carried out by the Laboratory of Bacteriology andVirology of the Pitie-Salpetriere hospital group directed by ProfessorJ. Grosset, it was shown that among the 709 AIDS patients whose bloodwas cultured (2460 samples) 63 (8.9%) had positive haemocultures withmycobacteria. On the other hand, 84.1% of the positive haemoculturescontained M. avium-intracellulare and 11.1% M. tuberculosis.

M. avium and M. intracellulare are two phenotypically related speciesindiscernible by conventional identification, and are therefore groupedinto an MAI complex. MAI complex for the purposes of the presentinvention is understood to mean the strains as defined by SAITO et al.(J. Clin. Microbiol., August 1990, 28, p. 1694-1697). This complexbrings together human and animal pathogens. In man non-infected by theHIV viruses, the infections are especially located in the lungs inadults and in the ganglia in children. MAI infections are diagnosed in50 to 90% of AIDS patients. The locations are, in this case, preferablyin the digestive system and are disseminated. MAI is also a pathogenwhich is important in veterinary medicine, affecting poultry, bovine,caprine and ovine species and the like. The animal pathogens, M.paratuberculosis and M. silvaticum, were recently introduced into thenomenclature as sub-species of M. avium (Thorel et al., Int. J. Syst.Bacteriol., 1990, 40, 254-260). It should also be noted that strains ofM. paratuberculosis were isolated from patients suffering from Crohn'sdisease.

Using serology, 28 serotypes are recognised within MAI. Currently,serotypes 1 to 6, 8 to 11 and 21 are classified as M. avium andserotypes 7, 12 to 20 and 25 as M. intracellulare. An uncertaintyremains as to the taxonomic position of serotypes 22 to 24 and 26 to 28.In man non-infected with the HIV viruses, the distribution of theserotypes is highly dispersed whereas in AIDS patients, only serotypes1, 4, 8 and 9 are predominant (data of the National Centre of Referencefor Mycobacteria). This distribution is similar to that observed inanimals where serotypes 1, 2, 3, 4, 8 are preferably found. The Frenchepidemiological situation for the M. avium strains isolated from AIDSpatients is different from that observed in the United States or inSweden. While in France serotype 8 is predominant, serotypes 4 or 6 arethe ones found in these other two countries.

The serotypes (or serovars) mentioned in the present patent are asdefined by P. J. Brennan et al. (Identification of A typicalMycobacteria by Thin-Layer Chromatography of Their Surface Antigens,Journal of Clinical Microbiology, Oct. 1978, p. 374-379.

Opportunistic mycobacteria pose numerous challenges to themicrobiologist, the immunologist and the clinician. Indeed, unlike M.tuberculosis which has a homogeneous bacterial population, the strainsof the M. avium-intracellulare complex are composed of cells which givevarious types of colonies on culture; a discrepancy was observed betweenthe response of patients to antituberculous chemotherapy and the resultsof the antibiogram in vitro, calling into question the role ofantibiograms (in any case, the MAI infections respond barely or poorlyto various antibiotic and antitubercolous treatments or the like);mycobacteria are responsible for the non-specific sensitization totuberculin and can also affect the immune response.

In the laboratory, the difficulty consists in isolating opportunisticmycobacteria from various samples and then in determining the species.The ubiquitous presence of opportunistic mycobacteria in the environmentoften renders the distinction between colonization and infectiondifficult. That is why it is important to refer to precise clinical andbacteriological criteria before deciding on the clinical significance ofa mycobacterinm isolated from a sample. Consequently, it is essential toestablish a cooperation of good quality between clinicians andbacteriologists and to have reliable and specific techniques foridentifying opportunistic mycobacteria. It is evident that themycobacterial species isolated and identified from pathological productsare linked to the isolation techniques and to the schemes used nowadaysfor their identification. Bacteriologists know that the identificationof opportunistic mycobacteria is made difficult by several elements:certain species are more or less sensitive to the decontaminationtechniques required before the culture, several species are isolated inprimary culture only under certain conditions, finally, certainidentification techniques based on phenotypic properties are notsufficiently reliable and can lead to a wrong identification. On theother hand, the taxonomic status of certain mycobacteria related to theMAI complex still remains uncertain and, in this case, theidentification cannot be established.

Mycobacteria of the M. avium-intracelllulare complex can, like M.tuberculosis, be responsible for adenites in children. The clinicianfrequently gives an initial antituberculous treatment while awaiting themicrobiological diagnosis which may take up to several weeks, thistreatment being stopped as soon as the microbiological diagnosis isobtained.

In order to avoid these errors of interpretation and to permit a rapiddiagnosis, the inventors responsible for the present invention setthemselves the objective of isolating DNA sequences specific formycobacteria belonging to the M. avium-intracellulare complex and ofdeveloping, using these sequences, a method of identification based onthe PCR technique and the molecular hybridization technique. Theytherefore searched for sequences which can be used both for thediagnosis and for the molecular epidemiology.

Recently, approaches using molecular hybridization to identify M.avium-intracellulare species have been proposed; however, most of thesemethods permit identification only after culture since they are notsufficiently sensitive to detect these mycobacteria directly in thebiological samples. This is the case for example for the cDNA probewhich is complementary to ribosomal RNA sequences and marketed byGene-Probe (San Diego, Calif.), see WO 84/02721, WO 88/03957, Musial, C.E. et al. (J. Clin. Microbiol., 1988, 26, 2120-2123). Furthermore,mycobacterial strains according to the criteria of Saito et al. (J.Clin. Microbiol, 1990, 28, 1694-1697) are not detectable using theprobes marketed by Gene-Probe. Other authors have found similar results:Enns, R. K. (Lab. Med. 1988, 19, 295-300), Sherman, I. et al. (J. Clin.Microbiol., 1989, 27, 241-244).

FR-2,645,878 describes, in addition, a nucleic sequence of 383 basepairs homologous in at least 5 mycobacteria species including M.tuberculosis.

This sequence has, however, the disadvantage of not being specific forthe M. avium-intracellulare complex.

A more sensitive and more specific method was therefore necessary inorder to establish a reliable diagnosis for atypical mycobacterioses anda precise identification of the various opportunistic mycobacteria.

SUMMARY OF THE INVENTION

The object of the present invention is to provide such a method as wellas the elements using this method.

The subject of the present invention is thus a nucleic acid sequencewhich hybridizes specifically with a genomic nucleic acid sequence ofthe strains belonging to the Mycobacterium avium-intracellulare complex,chosen from the nucleotide sequence SEQ ID No. 1, the nucleotidesequence SEQ ID No. 2, sequences complementary to these, as well as thesequences differing therefrom by mutation, insertion, deletion orsubstitution of one or more bases, and which does not hybridize with thenucleic acids of mycobacteria not belonging to the said complex.

The subject of the present invention is also a nucleic acid sequencechosen from the nucleotide sequences SEQ ID No. 1 and SEQ ID No. 2,their complementary sequences as well as the sequences differingtherefrom by mutation, insertion, deletion or substitution of one ormore bases and which have the same specificity.

The nucleic acid sequences defined above can be DNA sequences or RNAsequences.

The exact size of the fragment of sequence SEQ ID No. 2 (fragment I6) is1642 base pairs. The search in the EMBL and Genbank data banks revealedno significant homology with the DNA sequences already known. FragmentI6 was cloned into the plasmid pUC18 and the recombinant plasmid iscalled pMA01.

The plasmid pMA01 was deposited at the Collection nationals de Culturesde Microorganismes on 28 Aug. 1991 under the no. 1-1137.

The fragment of sequence SEQ ID No. 1 (fragment I1) used as probe on thedifferent M. avium-intracellulare serotypes shows a hybridization withserotypes 2, 3, 7, 12, 13, 14, 15, 16, 17, 18, 19, 20, 23, 24 and 25.Fragment I1 (SEQ ID No. 1) hybridizes with M. intra-cellulare strainsand the M. avium strains serotypes 2 and 3.

Nowadays, serovars 1, 2, 3, 4, 5, 6, 8, 9, 10, 11 and 21 are consideredas M. avium and serovars 7, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 25 asM. intracellulare.

Fragment I1 (SEQ ID No. 1), whose size is equal to 2004 base pairs, hasbeen cloned into the plasmid pUC18; the recombinant plasmid is calledpMA02.

The plasmid pMA02 was deposited at the Collection Nationals de Culturesde Microorganismes on 28 Aug. 1991 under the no. 1-1138.

These plasmids, as well as, more generally, a cloning vector containinga nucleic acid sequence as defined earlier, constitute another subjectof the invention.

An examination of the data bank reveals no homology between the sequenceof I1 (SEQ ID No. 1) and known sequences.

Fragments I6 (SEQ ID No. 2) and I1 (SEQ ID No. 1), functionallyequivalent portions or variants thereof, can be used in molecularhybridization techniques for the detection and identification of speciesof the M. avium-intracellulare complex. The functionally equivalentvariants comprise sequences in which base pairs have been mutated,deleted, inserted or substituted without the properties essential forthe specificity of these fragments being affected.

A nucleic acid fragment of 700 base pairs obtained by cleaving theinitial fragment I6 (SEQ ID No. 2) with the restriction enzymes SalI andEcoRI was used as probe on the DNA of the different serotpyes of M.avium. A hybridization is observed only with the DNA of the M. aviumstrains carrying the serotypes 1, 2, 3, 4, 5, 6, 8, 9, 10, 11 and 21. Itis important to note that this 700 base pair probe makes it possible todetect the M. avium serotypes most often encountered in AIDS patients.

The sequences I6 (SEQ ID No. 2) and I1 (SEQ ID No. 1) or the fragmentsobtained from these sequences can also be used to select primers for thePCR technique.

This technique necessitates the choice of oligonucleotide pairs flankingthe fragment which should be amplified (U.S. Pat. No. 4,683,202). Theseoligodeoxyribonucleotide or oligoribonucleotide primers haveadvantageously a length of between 18 and 30 and preferably 18 and 22nucleotides. One of the two primers is complementary to the (+) strandof the template and the other primer is complementary to the (-) strand.It is important that these primers do not possess a secondary structureor a mutually complementary sequence. On the other hand, the length andthe sequence of each primer should be chosen such that the primers donot hybridize with other nucleic acids obtained from prokaryotic oreukaryotic cells, in particular with the nucleic acids of mycobacterianot belonging to the M. avium-intracellulare complex and with human DNAor RNA which may possibly contaminate the sample.

The amplimers selected as specific primers for the amplification ofnucleic sequences of strains belonging to the MAI complex were chosenaccording to the method described by Griffais et al. (Nucleic Acids Res.1991, 19, 3887-3891).

From sequence I6 (SEQ ID No. 2), the inventors chose oligonucleotides inorder to perform a PCR test. Using these oligonucleotides, they obtainedan amplification specific for M. avium (serotypes 1, 2, 3, 4, 5, 6, 8,9, 10, 11 and 21) and for M. paratuberculosis, no amplifcation wasvisible with a nucleic acid of M. scrofulaceum, or of mcycobacteriabelonging to the tuberculosis complex.

Likewise, from sequence I1 (SEQ ID No. 1), the inventors selectedoligonucleotide primers which made it possible to develop a specific PCRtest. With the primers selected from fragment I1 (SEQ ID No. 1), theyobserved a fragment amplified only when the nucleic acid subjected toamplification came from the M. avium-intracellulare strains carryingserotypes 2, 3, 7, 12, 13, 14, 15, 16, 17, 18, 19, 20, 23, 24 and 25.

The preferred pairs of primers are represented in FIG. 1. Theoligonucleotide pairs which can be used as primers comprise preferablyat least 18 consecutive nucleotides chosen while conserving the 3' endof the sequences.

The most particularly preferred pair of primers are represented by thepairs consisting of oligonucleotides MA1 and MA2 derived from sequenceI6 (SEQ ID No. 2) on the one hand, and MA3 and MA4 derived from sequenceI1 (SEQ ID No. 1) on the other hand, of sequences: [SEQ ID NOS:

    ______________________________________                                        MA1:   .sup.5' GGC CCG ATG CGA CGT CGA GA.sup.3'                              MA2:   .sup.5' GCG CAA CTG CGC TTC GTC GA.sup.3'                              MA3:   .sup.5' GAA CGC CCG TTG CTG GCC ATT CAC GAG GAG.sup.3'                 MA4:   .sup.5' GCG CAA CAC GGT CGG ACA GGC CTT CCT CGA.sup.3'                 ______________________________________                                    

The amplified fragments can be identified after an agarose orpolyacrylamide gel electrophoresis or after a capillary electrophoresisor alternatively after a chromatographic technique (gel filtration,hydrophobic or ion-exchange chromatography). The specificity of theamplification can be controlled by molecular hybridization using, asprobes, the nucleic sequences I6 (SEQ ID No. 2) or I1 (SEQ ID No. 1),fragments thereof, plasmids containing these sequences or fragmentsthereof, oligonucleotides complementary to these sequences or fragmentsof sequences or products of amplification. These probes can be labelledor otherwise with radioactive elements or with non-radioactivemolecules.

These probes comprise advantageously at least 20 consecutive nucleotidesamong the sequences or fragments of sequences mentioned above, andconstitute another subject of the invention.

The probes are DNA probes or RNA probes.

The nucleic acid sequences described in this invention can thus be usedas probes to detect specifically and directly strains belonging to theM. avium-intracellulare complex in a biological sample. The non-labelledsequences can be used directly as probes, however, the nucleic acidsequences are generally labelled with a radioactive element (³² p, ³⁵ S,³ H, ¹²⁵ I) or with a non-radioactive molecule (biotin,acetylaminofluorene, digoxygenin, 5-bromodeoxyuridine) in order toobtain probes which can be used for numerous applications.

In this latter case, one of the labelling methods described in FR 2,422,956 and FR 2,518,755 can be used. The hybridization technique can becarried out in various ways (Matthews, J. A. and Kricka, L. J., Anal.Biochem. 1988, 169, 1-25). The method most widely used consists inimmobilizing the nucleic acid extracted from the mycobacterial cells ona support (nitrocellulose, nylon, polystyrene and the like) and inincubating, under well defined conditions, the immobilized targetnucleic acid with the probe nucleic acid. After hybridization, theexcess probe is removed and the hybrid molecules formed are detected bythe appropriate method (measurement of the radioactivity, thefluorescence or the enzymatic activity linked to the probe and thelike).

In another application, the nucleic acid probes described here can beused as capture probes. In this process, the probe is immobilized on asupport and is used to capture by specific hybridization the targetnucleic acid extracted from the mycobacteria. If necessary, the solidsupport is separated from the sample and the duplex formed between thecapture probe and the target nucleic acid is then detected by means of asecond detection probe labelled with an easily detectable element.

When a sufficient quantity of mycobacterial nucleic acid can beextracted from the samples to be analysed, the sequences described inthe patent can be used to detect and identify the strains belonging tothe M. avium-intracellulare complex directly in these samples. In theopposite case, a rapid culture in liquid medium can be carried outbefore extraction of the mycobacterial nucleic acid or, alternatively,the small quantity of mycobacterial nucleic acid extracted from thesample can be subjected to the PCR technique.

The subject of the present invention is also a method of detection ofthe presence of strains belonging to the Mycobacteriumavium-intracellulare complex in a biological sample, characterized bythe following steps:

i) bringing the biological sample into contact with a pair of nucleicacid fragments, called primers, as defined earlier, the nucleic acidcontained in the sample having been, where appropriate, previouslyrendered accessible to hybridization and under conditions permitting ahybridization of the primers to the nucleic acid of the strainsbelonging to the Mycobacterium avium-intracellulare complex;

ii) amplification of the nucleic acid of the strains belonging to theMycobacterium avium-intracellulare complex;

iii) visualization of the amplification of nucleic acid fragmentscorresponding to the fragment flanked by the primers;

iv) optional verification of the sequence of the amplified fragment, forexample by specific probe hybridization, by sequencing or by restrictionsite analysis.

The subject of the present invention is, in addition, a kit for thedetection of the presence of strains belonging to the Mycobacteriumavium-intracellulare complex in a biological sample, characterized inthat it comprises the following elements:

a pair of nucleic acid fragments as defined earlier;

the reagents necessary for carrying out an amplification of nucleic acidof strains belonging to the Mycobacterium avium-intracellulare complex;

optionally a component which makes it possible to verify the sequence ofthe amplified fragment, more particularly a nucleic probe as definedearlier.

This kit more advantageously contains the labelled or non-labelledprobe(s). These can be in solution or immobilized on a support. The kitmay also contain the reagents necessary for lysis of the mycobacteriaand extraction of the target nucleic acids, as well as the hybridizationand washing solutions corresponding to the chosen method.

The invention is illustrated in greater detail in the followingdescription, for which reference should be made to the accompanyingfigures in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the nucleotide sequences suitable for the choice ofoligonucleotide primers.

FIGS. 2A, 2B and 2C represent the Southern blot analysis of the cosmidDNA I6 (SEQ ID No. 2) of cosmid DNA, 15 μl of DNA approximatelycorresponding to 3 μg were digested with 10 units of enzyme SalI. Afterelectrophoresis, the DNA was transferred onto nitrocellulose andhybridized with the radioactive probes:

M. avium (A), M. intracellulare (B), M. tuberculosis (C). The arrowindicates the relevant fragment of 1642 base pairs.

FIGS. 3A, 3B and 3C represent the Southern blot analysis of the cosmidDNA I1 SEQ ID No. 1.

15 μl of cosmid DNA approximately corresponding to 3 μg were digestedwith 10 units of enzyme SalI. After electrophoresis, the DNA wastransferred onto nitrocellulose and hybridized with the radioactiveprobes: M. avium (A), M. intracellulare (B), M. tuberculosis (C). Thearrow indicates the relevant fragment of 2004 base pairs.

FIG. 4 represents the Southern blot analysis of the DNA of mycobacteriaof the M. avium-intracellulare complex.

2 μg of DNA were digested with 10 units of enzyme PstI. Afterelectrophoresis, the DNA was transferred onto nitrocellulose andhybridized with 25 ng of the radio-labelled SalI-EcoRI fragment derivedfrom I6 SEQ ID No. 2. This probe detects fragments in serotypes 1, 2, 3,4, 5, 6, 8, 9, 10, 11 and 21.

FIG. 5 represents the Southern blot analysis of the DNA of mycobacteriaof the M. avium-intracellulare complex.

2 μg of DNA were digested with 10 units of enzyme PstI. Afterelectrophoresis, the DNA was transferred onto nitrocellulose andhybridized with 25 ng of the radio-labelled SalI-BamHI fragment derivedfrom I1 SEQ ID No. 1. This probe detects fragments in serotypes 2, 3, 7,12, 13, 14, 15, 16, 17, 18, 19, 20, 23, 24 and 25.

FIG. 6 represents the agarose gel analysis of the amplified samples.

10 μl of the samples amplified with the pair of primers No. 1 describedin FIG. 1 and derived from the fragment I6 (SEQ ID No. 2) are depositedon a 2% agarose gel in TAE. The amplified fragments corresponding toserotypes 1, 2, 3, 4, 5, 6, 8, 9, 10, 11 and 21 are visualized under UVin the presence of ethidium bromide.

FIG. 7 represents the agarose gel analysis of the amplified samples.

10 μl of the samples amplified with the oligonucleotides MA3 and MA4derived from the fragment I1 SEQ ID No. 1 are deposited on a 2% agarosegel in TAE. The amplified fragments corresponding to serotypes 2, 3, 7,12, 13, 14, 15, 16, 17, 18, 19, 20, 23, 24 and 25 are visualized underUV in the presence of ethidium bromide.

FIGS. 8A and 8B represent the diagrams of the strategies followed forsequencing the fragments I6(A) SEQ ID No. 2 and I1 (SEQ ID No. 1) (B).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Construction of the M.avium Genomic Library

Screening of the Library and Determination of the Sequence of theSpecific Fragments

The genomic DNA of M. avium (serotype 2) is partially digested with therestriction endonuclease SalI by reacting 0.03 U of enzyme per μg of DNAin the buffer recommended by the supplier, for 1 hour at 37° C. Thegenomic DNA thus digested is separated by electrophoresis on 0.4%agarose gel. The fragments whose length is between 30 and 40 kb areelectroeluted and precipitated with ethanol after phenol/chloroform(1/1) extractions.

The vector is the cosmid pHC79. It is digested in the same manner anddephosphorylated in order to avoid any self-ligation.

The ligation is carried out by mixing 700 ng of vector, 0.8 μg of DNAfragments of 30/40 kb, the mixture is left at 14° C. for 18 h afterhaving added 2.5 units of T4 DNA ligase in an appropriate buffer.

The recombinant cosmids are packaged in vitro and used to transform thebacteria (HB101). The transformed bacteria are incubated for 1 h at 37°C. in LB medium and then plated on selective agar medium containing 25μg/ml of ampicillin. The ampicillin-resistant colonies are all testedfor their sensitivity to tetracycline, the DNA fragment of 30/40 kbhaving been inserted into the vector so as to inactivate thetetracycline resistance gene (Tet) and to conserve the ampicillinresistance gene (Amp).

A mini-preparation of DNA from the first 80 transformant coloniesresistant to ampicillin (Amp^(r)) and sensitive to tetracycline(Tet^(s)) is performed according to the alkaline lysis technique. TheDNA from these preparations is than digested with the restrictionendonuclease SalI, analysed by electrophoresis on 0.8% agarose gel andthen transferred onto nylon filters. The DNA is irreversibly bound byexposure for 5 min to UV at 254 nm.

These different filters are incubated for 16-18 hours at 68° C. in 6×SSC buffer (1× SSC corresponds to 0.15M NaCl and 0.015M Na citrate)containing 10% dextran sulphate, a 5× concentrated Denhardt solution (a1× Denhardt solution corresponds to 0.02% Ficoll, 0.02%polyvinylpyrrolidone and 0.02% bovine serum albumin), 10 mM EDTA, 0.5%SDS, 100 μg/ml of denatured salmon sperm DNA and genomic DNAradiolabelled with ³² phosphorus by multipriming, of one of thefollowing three species: M. avium, M. intracellulare, M. tuberculosisH37rv.

After hybridization, the filters are washed twice for 10 min in 2× SSCat 65° C., once for 30 min in 2× SSC supplemented with 0.1% SDS at 65°C. and finally once for 10 min in 0.1× SSC at 65° C. The filters, stillmoist, are subjected to autoradiography at -80° C. with an intensifyingscreen for 1 h to 2 days.

The results of these hybridizations made it possible to isolate twocosmid clones containing fragments of about 2 kb, called I6 SEQ ID No. 2and I1 (SEQ ID No. 1).

Fragment I6 hybridizes only with the genomic DNA of M. avium, fragmentI1 SEQ ID No. 1 hybridizes with the DNA of M. avium and M.intracellulare. Neither hybridizes with the labelled DNA of M.tuberculosis H37rv (FIGS. 2 and 3).

Fragments I6 SEQ ID No. 2 and I1 SEQ ID No. 1 were cloned into a vectorpUC18 and prepared in large quantities. The resulting plasmids werecalled pMA01 and pMA02 respectively.

These fragments were cleaved by the enzymes SmaI, BamHI, PstI and EcoRI,and then cloned into the phages M13mp18 and M13mp19 and sequencedaccording to the Sanger method using Taq polymerase in the presence ofd-azaGTP in place of dGTP. All the sequencing reactions were carried outwith ³⁵ S-labelled dATP.

The diagrams of FIG. 8 represent the strategies followed for thesequencing of fragments I6 SEQ ID No. 2 and I1 SEQ ID No. 1.

The entire sequences of the fragments are represented in the appendix(SEQ ID No. 2 and SEQ ID No. 1 corresponding to fragment I6 SEQ ID No. 2and fragment I1 SEQ ID No. 1 respectively). The composition between thewhole of the Los Alamos data bank and the 1642 nucleotides of fragmentI6 SEQ ID No. 2 and the 2004 nucleotides of fragment I1 SEQ ID No. 1,thus determined, reveal no significant homology with the sequencescurrently known.

The invention is illustrated by the following examples:

EXAMPLE 1

Analysis of mycobacterial DNA by the Southern technique using, asprobes, the nucleic acid sequences of the invention.

The list of mycobacteria used in this study is given in the followingtable:

                  TABLE                                                           ______________________________________                                        Names of the strains used                                                     SPECIES      STRAIN     CIPT* REFERENCE                                       ______________________________________                                        M. avium     ATCC* 25291                                                                              031 002                                               subsp.       ATCC 19698 112 0001                                              paratuberculosis                                                              M. intracellulare                                                                          ATCC 13950 031 0001                                              M. scrofulaceum                                                                            ATCC 19981 022 0001                                              M. tuberculosis                                                                            ATCC 27294 001 0001                                              M. bovis     ATCC 19210 002 0001                                              M. bovis BCG TMC*** 1011                                                                              004 0004                                              Pasteur                                                                       ______________________________________                                         *Collection Institut Pasteur Tuberculose                                      **American Type Culture Collection                                            ***Trudeau Mycobacterial Collection                                      

After culture on Lowenstein-Jensen or Middlebrook 7H9 media, themycobacteria are lysed using the following technique: the bacteria arecentrifuged for 30 minutes at 7000 g, resuspended in 20 ml of 7H9 mediumcontaining 100 μg/ml of D-cycloserine, 14 μg/ml of glycine, 200 μg/ml oflysozyme and 6 mM of EDTA and then incubated at 37° C. for 18 hours in 4ml of lysis buffer consisting of 50 mM Tris-HCl, pH 8, 50 mM EDTA, 100mM NaCl, 0.5% SDS and containing 60 μg/ml of pronase.

After the lysis, the DNAs are extracted with phenol-chloroform mixtureand precipitated with ethanol. The DNAs thus extracted are subjected toa total digestion with the enzyme PstI. The fragments obtained are thenseparated by electrophoresis on a 0.8% agarose gel in TAE before beingtransferred onto nitrocellulose membrane according to the Southerntechnique.

The fragments transferred are analysed by molecular hybridization. Inthis example, the probes used are the fragment of 700 base pairsextracted by digestion of fragment I6 SEQ ID No. 2 with the enzymes SalIand EcoRI and pMA02 which are labelled with ³² phosphorus according tothe technique of the "Multiprime" kit marketed by Amersham. The resultsare presented in FIGS. 4 and 5.

The probe derived from sequence I6 SEQ ID No. 2 detects two fragmentswith the DNAs of the M. avium strains (serotypes 1, 2, 3, 5, 6, 8, 9,10, 11 and 21) and only one fragment with the DNAs of the straincarrying serotype 4. The size of these fragments varies with the straintested, different patterns are thus obtained by Southern blotting (FIG.4).

The probe pMA02, containing sequence I1 SEQ ID No. 2, detects a singlefragment on the DNAs of the M. avium-intracellulare strains (serotypes2, 3, 7, 12, 13, 14, 15, 16, 17, 18, 19, 20, 23, 24 and 25). The size ofthe fragment obtained is variable and is between about 5 and 20 kb (FIG.5).

EXAMPLE 2

Enzymatic amplification in vitro of the DNA of M. avium-intracellularestrains with the primers defined from the nucleic acid sequences whichare the subject of the invention.

Synthesis of the Oligonucleotide Primers

The primers derived from sequences I6 SEQ ID No. 2 and I1 SEQ ID No. 1and having a length of between 18 and 22 nucleotides were synthesized inan Applied Biosystem automated apparatus based on the chemistry ofphosphoramidites. After the synthesis, the oligonucleotide solution istransferred into a glass tube and incubated with concentrated ammoniumhydroxide at 50° C. for 4 hours. The ammonium hydroxide is thenevaporated in a Speed Vac/Concentrator and the pellet is washed twicewith sterile distilled water and dried. Finally, the pellet is taken upin 0.5 ml of Tris-HCl buffer, pH 8.0 containing 1 mM EDTA. Theconcentration of each primer is determined using a spectrophotomer andan aliquot is analysed by electrophoresis on a polyacrylamide gel inorder to verify that the primer is not degraded.

Isolation of the Mycobacterial DNA

The biological extracts are treated in an appropriate manner and thencentrifuged. The DNA is extracted by resuspending the pellet with 50 μlof 0.1M NaOH containing 2M NaCl and 0.5% SDS. The mixture is incubatedat 95° C. for 15 minutes; 400 μl of 0.1M Tris-HCl, pH 7, are added tothe reaction mixture. The DNA is extracted 3 times withphenol/chloroform, precipitated with ethanol and taken up in 50 μl of 10mM Tris buffer, pH 8, containing 0.1 mM EDTA.

Amplification

The enzymatic amplification technique in vitro (PCR) is carried outaccording to the procedure described by Saiki et al. (Science, 1988,239, 487-491) using 12.5 pmol of the oligonucleotides of the pair ofprimers No. 1 of FIG. 1, on the one hand, or MA3 and MA4, on the otherhand, and 5 ng of DNA of different strains of mycobacteria with 2 unitsof Taq polymerase in a buffer containing 50 mM KCl, 10 mM Tris-HCl, pH8.3, 2 mM MgCl₂, 300 μM of deoxyribonucleotide triphosphates and 100μg/ml of gelatin, the final volume of the reaction being 100 μl. Theparameters for the PCR stages were chosen in the following manner: 2 minat 94° C., 2 min at 50° C., 2 min at 72° C. Thirty cycles are carriedout using an automated apparatus. After the last cycle, the samples aremaintained at 4° C. until the analysis.

Electrophoretic Analysis on an Agarose Gel of the Amplified Samples

Ten μl of the samples are loaded onto a 2% agarose gel in a TAE buffer(0.04M Tris-acetate, 0.001M EDTA) containing 1 μg/ml of ethidiumbromide. The amplified fragments are visualized under UV and the gelsare photographed using a Polaroid 667 film.

FIGS. 6 and 7 show the results obtained with different mycobacteria DNAsand the pair of primers No. 1, on the one hand (FIG. 6), and with theprimers MA3 and MA4 on the other hand (FIG. 7).

When the pair of primers No. 1 is used, a DNA fragment corresponding tothe expected size is observed with the DNAs of the followingmycobacteria: M. avium (serotypes 1, 2, 3, 4, 5, 6, 8, 9, 10, 11 and 21)and M. paratuberculosis.

It is important to note that the pair of primers No. 1 makes it possibleto detect M. paratuberculosis, making the method usable in the field ofveterinary medicine. Indeed, M. paratuberculosis is responsible forJohne's disease in ruminants. This diseases poses an economic threat; itresults in a loss of production of meat and milk of which the cost isestimated, in the United States of America, at one billion dollars peryear.

When the pair of primers MA3 and MA4 is used, a DNA fragmentcorresponding to the expected size is observed with the DNAs of thefollowing mycobacteria: M. avium (serotypes 2, 3, 7, 12, 13, 14, 15, 16,17, 18, 19, 20, 23, 24 and 25).

On the other hand, no amplified fragment is visible when the DNAanalysed is extracted from the following strains: M. tuberculosis, M.boris, M. bovis-BCG, M. microti, M. scrofulaceum, M. fortuitum, M.kansasii and when one of the pairs of primers No. 1, the pair MA1 andMA2 or the pair MA3 and MA4 is used to direct the amplification.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 66                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2004 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTCGACGCCACCACACTGCCCCACGACATCGAACGTCCTGGCCGGCACGATCGCGAAGGC60                GGGAACGGTTGTCGGGCAGCGAATTCTCGTGGGTCGGCCACTGGTCGGGAACGCCCGTTG120               GCTGGCCATTCACGAAGGAGTGGGTGCTCACCCGCGAACCTTCCACAATGGGGCATGGCT180               CCATTGGCGCCCGGCGAAAAGGACGCCGCTGATCCGCGCGGTCATCAAAGGTGAGCCCAG240               CTTTGAACTCCAGCTCGACGTGGCATTCGACGGCGCGCCATCGAACGGGCCAGCACGCCA300               TGCCAGGTCACCTGATGATCGCGAATGAAGCGCGGTTCGCGCCATACCGTACGTGCTGGC360               CCGGCCACCCGGTGTCGTGACAGCACCGGTGTTCGGCGCGATCCAACTAGCCTGAGGCAC420               CACCGACCGCGCGGGCGATGTGGTTCGCTGGGCGCCGCATGGAAAACGTGCGCGCTGCCG480               TCGGGCAAAACCTTCGGGCCACGAGATTAATCGGAACCCATCCACCCCTGTCGGATGAAC540               CGGTCCGAATTCGCAGGTAACGTTCCCGGCGCGCCTGCTGGCCGACGGGAACGAGCCTTT600               CACCTGCTCCATTCCCGTTCTTCACACCCTCCCCGGTTCAACGGCCGTGCCGCGGCGAGA660               CCACGCACGATCACGGTGGCCGCGTCGTGCGACAGGCCCGGCATCGAGTGTCCGGGCCGG720               CGACCGTATCGCGCCTCGAAGCGGTCGAGGAAGGCCTGTCCGACCGTGTTGCGCTCGTCG780               TAGCTGTCCAGGCCGATCCATCCGGATAGGTGCCGCCTCCACTCCGCGCTGATGTGTGCC840               ATTTCGAACGCCGTCGTCGTGTATCGCGGCGGATCCCAGCCCGCGGCGCGCAGCGCATCC900               GTGAAGCCCCACAGCCCGTGCCCGAACCCGAACGTGCACCAGCCGTCGGGTTCCGCCGCA960               CGCAACGCAGTGACCGTCTCGGACTTGTCGGCTTCGACCTGGGGAATCGCCACGGTCGCA1020              ACGACCTTCAAACCCGCGGCGTCGTAGGCTCTCTCGGCGAAGGCGAGGTACCTCCTTACC1080              GATGAGCGATGCCTCGTAGCAATGCGATGCGCGGATCGACCGTCGCCGGATCATCACCGC1140              GGCCAGCATCACCGGCTCCTCGGGCATCGAGCCGTTGTTCAGTCGAAGCACCAGTCGCCG1200              AGCGCTCCCTCCGATCCCGACAGAGTGACGATCGGAACCCGGCGCGGTGGCCTCGACGTG1260              GGAACGCAGCGGCACCACGTTATCCGACACCCACGGGCCCGAAGATCGCCAGGCAACCCT1320              CCGCGACGAGCTCGTCGAAGGCCCGCTCCGACCACTTGGTAGGTCCCGTTGGGCAGGCCC1380              ACCACATTGCGGGTGACGAGTTCGATCGGCCGGTCGATCAGTCCCGAGGAAAGGGCTTCA1440              TCCATCACCAGCCGCAGCGCCGACGCTGTCGTTGTCGGTGTCGCCGGACGTCGGATAGTC1500              GTTGAGCAAGCCGACTTTCAGGGGAGCGACCGAACCGTATGCGCGAACCTCGTCTGCCAT1560              AGCGGCAACATACAGATATGGAGGTTGAGGCCCACCGCGCCACCGCAAACTCGCACCCGC1620              CGAAAATGGTGGTTTACGAAAAGTGCAAAGCGAACTTTCGGCATGTTAGTTTTTCCGAGC1680              GGGGGATCACATTCCGGAGGAGACGGAATGAACGCTCACATCGACGCAGGAGTCCCACGC1740              GCCGTCAATCCAGAAGAAGTACCGCGTGATCCAGTGGGGCATGGGTAATGTCGGCACGAT1800              CGCACTGCGCCACTTCGCACACAACCCTCTCTACGAAGTGGTCGGAGTGCTTTGTAATCG1860              CCCGGAGAAGGTCGGTAGGGAACGCCGGTGAACTTCGTCGGGGTAGGACCGGATCGGGGT1920              ACACGCAACCACCGATAAGGCCACCTTGGAGACACTCGACAGCCGATTGCGTGTTCTACG1980              CGCCGCTCTGGTCCGGACGTCGAC2004                                                  (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1642 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      GACCAGTTCCGCGTGCATTCGCCCGCCCACCTCGCCGACCAGCGAGGCCGTGTGGTCGAG60                ATCGTCGGGTGTGCTGGGCGGAACCATCACCGTGTCCACCGGTCCGCACAGGTCGAGGGC120               CACGATGTCGGCGCCCTCCTGCGCGAACCGCACGGCTTGGGCGCGCCCGATTCCCCGCGC180               CGCGCCCGTGATCAACGCGACCTTCCCCGCCAGCCGCCCCGGACTGACTTGTGGCATCCG240               GCGATTCTGCCGCGCGAGCGGCACCGGGGTCGGTGTGTGTCCGATGGCCGGGAGACGATC300               TATGCCGGCGTACCGGTCACCGGGATATCCCGGCCGAGGGCGACCGCCGGGACCTAACGT360               AGGCATCCGTGACCAGTGGCAGTGACCGGCGCGCAACCCCGGACGTCGATGTGGTGGTCG420               TCGGCGCGGCTTCGCCGGCCTGTACGCGCTGCACAAACTGCGATCGAACGGCTGCGGGTC480               AGGGTGTTCGAGGCCGGGCCGGACGTCGGCGGAACCTGGTACTTCAACCGCTACCCCGGG540               GCCCGATGCGACGTCGAGAGCGTGGACTACTGCTACTCGTTCTCCGACGCGCTGCAGCGG600               GAGTGGGATTGGTCGGAGAAGTACGCCACCCAACCCGAGATTCTGGCCTACATCAATTGG660               GTCGCCGACCGGCTGGATCTGCGCCGCGACATCACCCTGAACGCCCGCGTGAATTCCGCC720               GTGCTCGACGAAGCGCAGTTGCGCTGGACGGTGACCACCGAGACGGGCGAGCGCGTCACC780               GCCCGGTTCTGCGTGATGGCCACCGGCCCGTTGTCGGCGGCGATGACTCCGCCGTTCCCC840               GGTTTGGATACCTTCGCCGGGCAGGTCTACCACACCGCGGCCTGGCCGCACGAACGGTGG900               ACTTCACCGGCAAGCGCGTCGCCGTCATTGGCACGGGATCCCCGGGTCTGGCATCCAATG960               CAGATTCGCGATCACTCGACCGAGGCCGCATCGCAGCTGTACGTGTTCCAGCGCACCCCG1020              AATTACAGTGTGCCGGCGGGTAACCGGCCGTTGAGTGACTCCGACCGTGCCGAGGTGAAG1080              GCCCATTACGCGGAACGACGACGGATATCGTGGCGCACGGGGGGCGGCTCGCCCACGTCG1140              CCCACCCGAAGTTGACGATGGAGGCAACCCCCGAGGAACGGCGTGAGGCATTCGAAAAGC1200              GCTGGGAGCTCGGCGAGTGCTGTTCTCCAAGACATTCGCCGACCAGATGATCGACCCGGT1260              CGCCAACGAGGAGGCCCGCAAGTTCTACGAGGAGAAGGTGCGCGCGGTCATCGACGACCC1320              GGTGCTGGCCGACCTGCTCATCCCGAATGACCATCCGATCGGCACCAAGCGAATCTGCAC1380              CGACAGCAACTACTTTCAGACCTTCAACCGCCCGAACGTGAAGCTGATCAGCGTCCGCAA1440              GACGCCGATCATGTCCATCGATGCGACCGGCATTAACACCACCGACGCCCATTACGACCT1500              CGACGCGATCGTGCTGGCGACCGGATTCGACGCGATGACCGGCGCGCTGGCCAAGATCGA1560              CATCGTGGGCCGCGACGGAAGGCGGCTGAGCGACGACTGGTCCGGCGGGCCCCGCACGTA1620              CCTGGGCCTCGGCGTCGACCTG1642                                                    (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      ATGGCCGGGAGACGATCTATGCCGGCGTAC30                                              (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      CGTTCGATCGCAGTTTGTGCAGCGCGTACA30                                              (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      AACACCCTGACCCGCAGCCGTTCGATCGCA30                                              (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      GAACACCCTGACCCGCAGCCGTTCGATCGC30                                              (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      CGAACACCCTGACCCGCAGCCGTTCGATCG30                                              (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      GCCGGGAGACGATCTATGCCGGCGTACCGG30                                              (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      CCGGCCGAGGGCGACCGCCGGGACCTAACG30                                              (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     TCAACCGCTACCCCGGGGCCCGATGCGACG30                                              (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     TCACCGTCCAGCGCAACTGCGCTTCGTCGA30                                              (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     GCTACCCCGGGGCCCGATGCGACGTCGAGA30                                              (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                     GCCCGCGTGAATTCCGCCGTGCTCGACGAA30                                              (2) INFORMATION FOR SEQ ID NO: 14:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                     GACGCGCTTGCCGGTGAAGTCCACCGTTCG30                                              (2) INFORMATION FOR SEQ ID NO: 15:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                     GGATCCCTGCCAATGACGGCGACGCGCTTG30                                              (2) INFORMATION FOR SEQ ID NO: 16:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                     GATGCGGCCTCGGTCGAGTGATCGCGAATC30                                              (2) INFORMATION FOR SEQ ID NO: 17:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                     CGATGCGGCCTCGGTCGAGTGATCGCGAAT30                                              (2) INFORMATION FOR SEQ ID NO: 18:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                     GCGATGCGGCCTCGGTCGAGTGATCGCGAA30                                              (2) INFORMATION FOR SEQ ID NO: 19:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                     TGCGATGCGGCCTCGGTCGAGTGATCGCGA30                                              (2) INFORMATION FOR SEQ ID NO: 20:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                     CACGTACAGCTGCGATGCGGCCTCGGTCGA30                                              (2) INFORMATION FOR SEQ ID NO: 21:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                     ACCACACCGCGGCCTGGCCGCACGAACGGT30                                              (2) INFORMATION FOR SEQ ID NO: 22:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                     GCCACGATATCCGTCGTCGTTCCGCGTAAT30                                              (2) INFORMATION FOR SEQ ID NO: 23:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:                                     ACGAACGGTGGACTTCACCGGCAACGCCGT30                                              (2) INFORMATION FOR SEQ ID NO: 24:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:                                     CCCGGGTCTGGCATCCAATCGAGATTCGCG30                                              (2) INFORMATION FOR SEQ ID NO: 25:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:                                     CCGGGTCTGGCATCCAATGCAGATTCGCGA30                                              (2) INFORMATION FOR SEQ ID NO: 26:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:                                     CGGGTCTGGCATCCAATGCAGATTCGCGAT30                                              (2) INFORMATION FOR SEQ ID NO: 27:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:                                     GGGTCTGGCATCCAATGCAGATTCGCGATC30                                              (2) INFORMATION FOR SEQ ID NO: 28:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:                                     TCCAATGCAGATTCGCATCACTCGACCGAG30                                              (2) INFORMATION FOR SEQ ID NO: 29:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:                                     CGGCATCGAGTGTCCGGGCCGGCGACCGTA30                                              (2) INFORMATION FOR SEQ ID NO: 30:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:                                     ATCCGCCGCGATACACGACGACGGCGTTCG30                                              (2) INFORMATION FOR SEQ ID NO: 31:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:                                     GCGCCGCGGGCTGGGATCCGCCGCGATACA30                                              (2) INFORMATION FOR SEQ ID NO: 32:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:                                     CGCGCCGCGGGCTGGGATCCGCCGCGATAC30                                              (2) INFORMATION FOR SEQ ID NO: 33:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:                                     GCGCGCCGCGGGCTGGGATCCGCCGCGATA30                                              (2) INFORMATION FOR SEQ ID NO: 34:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:                                     CATCGAGTGTCCGGGCCGGCGACCGTATCG30                                              (2) INFORMATION FOR SEQ ID NO: 35:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:                                     ATCGAGTGTCCGGGCCGGCGACCGTATCGC30                                              (2) INFORMATION FOR SEQ ID NO: 36:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:                                     TCGAGTGTCCGGGCCGGCGACCGTATCGCG30                                              (2) INFORMATION FOR SEQ ID NO: 37:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:                                     CGAGTGTCCGGGCCGGCGACCGTATCGGCG30                                              (2) INFORMATION FOR SEQ ID NO: 38:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:                                     CGCAACGACCTTCAAACCCGCGGCGTCGTA30                                              (2) INFORMATION FOR SEQ ID NO: 39:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:                                     TCGAGGCCACCGCGCCGGGTTCCGATCGTC30                                              (2) INFORMATION FOR SEQ ID NO: 40:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:                                     GTCGAGGCCACCGCGCCGGGTTCCGATCGT30                                              (2) INFORMATION FOR SEQ ID NO: 41:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:                                     TGCTCTGTAGCAATGCGATGCGCGGATCGA30                                              (2) INFORMATION FOR SEQ ID NO: 42:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:                                     TCGTAGCAATGCGATGCGCGGATCGACCGT30                                              (2) INFORMATION FOR SEQ ID NO: 43:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:                                     GTAGCAATGCGATGCGCGGATCGACCGTCG30                                              (2) INFORMATION FOR SEQ ID NO: 44:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:                                     CCACCACATTGCGGGTGACGAGTTCGATCG30                                              (2) INFORMATION FOR SEQ ID NO: 45:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:                                     TATGGCAGACGAGGTTCGCGCATACGGTTC30                                              (2) INFORMATION FOR SEQ ID NO: 46:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:                                     TCCCCTGAAAGTCGGCTTGCTCAACGACTA30                                              (2) INFORMATION FOR SEQ ID NO: 47:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:                                     CTATGGCAGACGAGGTTCGCGCATACGGTT30                                              (2) INFORMATION FOR SEQ ID NO: 48:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:                                     CCGCTATGGCAGACGAGGTTCGCGCATACG30                                              (2) INFORMATION FOR SEQ ID NO: 49:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:                                     TGCCGCTATGGCAGACGAGGTTCGCGCATA30                                              (2) INFORMATION FOR SEQ ID NO: 50:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:                                     CACCACATTGCGGGTGACGAGTTCGATCGG30                                              (2) INFORMATION FOR SEQ ID NO: 51:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:                                     CTGTCGTTGTCGGTGTCGCCGGACGTCGAA30                                              (2) INFORMATION FOR SEQ ID NO: 52:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:                                     GATTGACGGCGCGTGGGACTCCTGCGTCGA30                                              (2) INFORMATION FOR SEQ ID NO: 53:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:                                     TGTCGTTGTCGGTGTCGCCGGACGTCGGAT30                                              (2) INFORMATION FOR SEQ ID NO: 54:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:                                     GTCGGTGTCGCCGGACGTCGGATAGTCGTT30                                              (2) INFORMATION FOR SEQ ID NO: 55:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:                                     GCCGACTTTCAGGGGAGCGACCGAACCGTA30                                              (2) INFORMATION FOR SEQ ID NO: 56:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:                                     AAGTGGCGCAGTGCGATCGTGCCGACATTA30                                              (2) INFORMATION FOR SEQ ID NO: 57:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:                                     CCGACTTTCAGGGGAGCGACCGAACCGTAT30                                              (2) INFORMATION FOR SEQ ID NO: 58:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:                                     ACTTTCAGGGGAGCGACCGAACCGTATGCG30                                              (2) INFORMATION FOR SEQ ID NO: 59:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:                                     TTTCAGGGGAGCGACCGAACCGTATGCGCG30                                              (2) INFORMATION FOR SEQ ID NO: 60:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:                                     GGAGACGGAATGAACGCTCACATCGACGCA30                                              (2) INFORMATION FOR SEQ ID NO: 61:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:                                     CGTTCCCTACCGACCTTCTCCGGGCGATTA30                                              (2) INFORMATION FOR SEQ ID NO: 62:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:                                     GGAGACGGAATGAACGCTCACATCGACGCA30                                              (2) INFORMATION FOR SEQ ID NO: 63:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:                                     GGCCCGATGCGACGTCGAGA20                                                        (2) INFORMATION FOR SEQ ID NO: 64:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:                                     GCGCAACTGCGCTTCGTCGA20                                                        (2) INFORMATION FOR SEQ ID NO: 65:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:                                     GAACGCCCGTTGCTGGCCATTCACGAGGAG30                                              (2) INFORMATION FOR SEQ ID NO: 66:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mycobacterium avium                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:                                     GCGCAACACGGTCGGACAGGCCTTCCTCGA30                                              __________________________________________________________________________

We claim:
 1. A purified nucleic acid sequence selected from the groupconsisting of the nucleotide sequence SEQ ID No. 1, the nucleotidesequence SEQ ID No. 2, sequences complementary to the nucleotidesequences of SEQ ID No. 1 or SEQ ID No. 2, and fragments of SEQ ID No. 1or SEQ ID No. 2 which specifically hybridize to only a genomic nucleicacid sequence of the Mycobacterium avium-intracellulare complex.
 2. Apurified nucleic acid sequence which hybridizes specifically to only agenomic nucleic acid of the serotypes 2, 3, 12, 13, 14, 15, 16, 17, 18,19, 20, 23, 24 and 25, of the M. avium-intracellulare complex comprisingthe nucleotide sequence SEQ ID No. 1, a sequence complementary thereto,and fragments thereof which specifically hybridize to only a genomicnucleic acid sequence of the serotypes 2, 3, 12, 13, 14, 15, 16, 17, 18,19, 20, 23, 24, and 25 of the Mycobacterium avium-intracellularecomplex.
 3. A purified nucleic acid sequence which hybridizesspecifically with only a genomic nucleic acid sequence of the M. aviumserotypes 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, or 21, said nucleic acidsequence comprising a nucleic acid fragment of 700 base pairs which isobtained by cleaving a nucleic acid having the nucleotide sequence SEQID No. 2 with SalI and EcoRI.
 4. An oligonucleotide primer pair specificfor the amplification of a nucleic sequence of the strains belonging tothe Mycobacterium avium-intracellulare complex, comprising a pair ofoligonucleotides having 18 to 30 nucleotides whose sequences areselected from the group consisting of the sequences according toclaim
 1. 5. An oligonucleotide primer pair according to claim 4comprising a pair of oligonucleotides having 18 to 22 nucleotides.
 6. Anoligonucleotide primer pair selected from the group consisting of theoligonucleotide pair MA1 and MA2 having the sequences:

    ______________________________________                                        MA1: .sup.5' GGC CCG ATG CGA CGT CGA GA.sup.3'  SEQ ID No. 63 and             MA2: .sup.5' GCG CAA CTG CGC TTC GTC GA.sup.3'  SEQ ID No. 64; and the             oligonucleotide pair MA3 and MA4 having the [of] sequences:              MA3: .sup.5' GAA CGC CCG TTG CTG GCC ATT CAC GAG GAG.sup.3'                        SEQ ID NO. 65 and                                                        MA4: .sup.5' GCG CAA CAC GGT CGG ACA GGC CTT CCT CGA.sup.3'  [.]                   SEQ ID No.
 66.                                                           ______________________________________                                    


7. A cloning vector comprising a nucleic acid sequence according toclaim
 1. 8. Plasmid pMA01 deposited at the CNCM on 28 Aug. 1991 underthe no. I-1137.
 9. Plasmid pMA02 deposited at the CNCM on 28 Aug. 1991under the no. I-1138.
 10. Oligonucleotide probes specific for a nucleicacid sequence of the strains belonging to the Mycobacteriumavium-intracellulare complex comprising at least 20 consecutivenucleotides selected from the group consisting of the nucleic sequencesaccording to claim
 1. 11. Oligonucleotide probes specific for a nucleicacid sequence of the strains belonging to the Mycobacteriumavium-intracellulare complex comprising the nucleotide sequences SEQ IDNo. 1, SEQ ID No. 2, fragments of these sequences, plasmids containingthe nucleotide sequences SEQ ID No. 1, SEQ ID No. 2 or fragments ofthese sequences, oligonucleotides complementary to nucleic acidsequences SEQ ID No. 1, SEQ ID No. 2 or to fragments of these sequences.12. Oligonucleotide probes according to claim 11 which are immobilizedon a support and are used as capture probes.
 13. A method of detectionof the presence of strains belonging to the Mycobacteriumavium-intracellulare complex in a biological sample comprising the stepsof:i) contacting the biological sample with a pair of primers accordingto claim 5 under hybridization conditions such that the primersspecifically hybridize with a nucleic acid sequence from a strainbelonging to the Mycobacterium avium-intracellulare complex; ii)amplifying the nucleic acid sequence which has hybridized with theprimer pair in the contacting step i); iii) visualizing the amplifiednucleic acid to thereby detect the presence of the strains belonging tothe Mycobacterium avium-intracellulare complex; and iv) optionallyverifying the sequence of the amplified nucleic acid.
 14. A kit for thedetection of the presence of strains belonging to the Mycobacteriumavium-intracellulare complex in a biological sample comprising:anoligonucleotide primer pair according to claim 4; reagents necessary forcarrying out an amplification of a nucleic acid of strains belonging tothe Mycobacterium avium-intracellulare complex; optionally a componentwhich makes it possible to verify the detection of the presence ofstrains belonging to the Mycobacterium avium-intracellulare complex. 15.The method according to claim 13 wherein the detection of the presenceof strains belonging to the Mycobacterium avium-intracellulare complexis verified by specific probe hybridization, by sequencing or byrestriction site analysis.
 16. The kit according to claim 14 whereinsaid component which makes it possible to verify the detection of thepresence of strains belonging to the Mycobacterium avium-intracellularecomplex is an oligonucleotide probe specific for a nucleic acid sequenceof the strains belonging to the Mycobacterium avium-intracellularecomplex comprising at least 20 consecutive nucleotides from a nucleicacid sequence which hybridizes specifically with a genomic nucleic acidsequence of the strains belonging to the Mycobacteriumavium-intracellulare complex, said nucleic acid sequence being selectedfrom the group consisting of the nucleotide sequence SEQ ID No. 1, thenucleotide sequence SEQ ID No. 2, sequences complementary to these, andfragments thereof which specifically hybridize to only the Mycobacteriumavium-intracellulare complex.
 17. An oligonucleotide primer pairaccording to claim 4 comprising a pair of oligonucleotides whosesequences are selected from the group consisting of the nucleotidesequences represented in FIG. 1.